In vitro Assays to Evaluate Specificity and Affinity in Protein-phospholipid Interactions

Hendry Susila, Snjezana Juric, Katarzyna Gawarecka, Kyung Sook Chung, Suhyun Jin, Soo Jin Kim, Zeeshan Nasim, Geummin Youn, Ji Hoon Ahn

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)


Protein-lipid interactions play important roles in many biological processes, including metabolism, signaling, and transport; however, computational and structural analyses often fail to predict such interactions, and determining which lipids participate in these interactions remains challenging. In vitro assays to assess the physical interaction between a protein of interest and a panel of phospholipids provide crucial information for predicting the functionality of these interactions in vivo. In this protocol, which we developed in the context of evaluating protein-lipid binding of the Arabidopsis thaliana florigen FLOWERING LOCUS T, we describe four independent in vitro experiments to determine the interaction of a protein with phospholipids: lipid-protein overlay assays, liposome binding assays, biotin-phospholipid pull-down assays, and fluorescence polarization assays. These complementary assays allow the researcher to test whether the protein of interest interacts with lipids in the test panel, identify the relevant lipids, and assess the strength of the interaction.

Original languageEnglish
Article numbere4421
Issue number10
Publication statusPublished - 2022 May 20

Bibliographical note

Funding Information:
This work was supported by a National Research Foundation (NRF) of Korea grant funded by the Korean government (NRF-2017R1A2B3009624 to J.H.A.) and Samsung Science and Technology Foundation (SSTF-BA1602-12 to J.H.A). The authors thank H.K. Song for fluorescence polarization spectroscopy experiments.

Publisher Copyright:
© 2022 Bio-protocol LLC. All Rights Reserved.


  • Fluorescence polarization
  • In vitro assay
  • Liposome
  • Phosphatidylcholine
  • Phosphatidylglycerol
  • Phospholipids
  • Protein-phospholipids interaction

ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)
  • Plant Science


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